Plasmids, Oligonucleotides, siRNAs and Antibodies

Table 1. Characteristics of plasmids used for site-directed mutagenesis and cellular transfection assays.

Plasmid Description Reference

pEYFP-N1 MCS at N-terminus of EYFP tag Clontech pEYFP-C1 MCS at C-terminus of EYFP tag Clontech pECFP-N1 MCS at N-terminus of ECFP tag Clontech pECFP-C1 MCS at C-terminus of ECFP tag Clontech

pECFP-EYFP ECFP and EYFP fusion construct Kind gift from Prof. Dr. Michael Schindler – University Hospital Tubingen (Banning et al., 2010) pEYFP-Survivin-N1 Survivin at N-terminus of EYFP

tag

Provided by Melanie Hoffmann – Master study - University Hospital Frankfurt (Hoffmann, 2017) pEYFP-Survivin-C1 Survivin at C-terminus of EYFP tag Provided by Melanie Hoffmann –

Master study - University Hospital Frankfurt (Hoffmann, 2017) pECFP-Survivin-N1 Survivin at N-terminus of ECFP

tag

Provided by Melanie Hoffmann – Master study - University Hospital Frankfurt (Hoffmann, 2017) pECFP-Survivin-C1 Survivin at C-terminus of ECFP tag Provided by Melanie Hoffmann –

Master study - University Hospital Frankfurt (Hoffmann, 2017) pEYFP-Survivin S20A-N1 S20A mutated Survivin at

N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin S20D-N1 S20D mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin W25A-N1 W25A mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin F27A-N1 F27A mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin L28A-N1 L28A mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin E29A-N1 E29A mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin C31A-N1 C31A mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin T34A-N1 T34A mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin T34D-N1 T34D mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin D53A-N1 D53A mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin W67A-N1 W67A mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin E76A-N1 E76A mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin K79A-N1 K79A mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin T117A-N1

T117A mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin T117D-N1

T117D mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin S20D-W67A-N1

S20D and W67A mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin S20D-C31A-D53A-N1

S20D, C31A, and D53A mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin C31A-D53A-N1

C31A and D53A mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin C31A-D53A-W67A-N1

C31A, D53A, and W67A mutated Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-Survivin ∆BIR-N1 BIR domain deleted Survivin at N-terminus of EYFP tag

Generated in this study

pEYFP-PI3K-N1 PI3K domain of DNA-PKcs at N-terminus of EYFP tag

Provided by Melanie Hoffmann – Master study - University Hospital Frankfurt (Hoffmann, 2017) pEYFP-PI3K-C1 PI3K domain of DNA-PKcs at

C-terminus of EYFP tag

Provided by Melanie Hoffmann – Master study - University Hospital Frankfurt (Hoffmann, 2017) pECFP-PI3K-N1 PI3K domain of DNA-PKcs at

N-terminus of ECFP tag

Provided by Melanie Hoffmann – Master study - University Hospital Frankfurt (Hoffmann, 2017) pECFP-PI3K-C1 PI3K domain of DNA-PKcs at

C-terminus of ECFP tag

Provided by Melanie Hoffmann – Master study - University Hospital Frankfurt (Hoffmann, 2017) pEYFP-HEAT1-N1 HEAT1 repeat of DNA-PKcs at

N-terminus of EYFP tag

Provided by Melanie Hoffmann – Master study - University Hospital Frankfurt (Hoffmann, 2017) pEYFP-FATC-N1 FATC domain of DNA-PKcs at

N-terminus of EYFP tag

Provided by Melanie Hoffmann – Master study - University Hospital Frankfurt (Hoffmann, 2017) pEYFP-PI3K-ATM-N1 PI3K domain of ATM at

N-terminus of EYFP tag

Provided by Melanie Hoffmann – Master study - University Hospital Frankfurt (Hoffmann, 2017) p3xFlag-CMV10-Survivin Survivin at C-terminus of 3xFlag

tag

Sigma Aldrich - Generated in this study by inserting the Survivin to EcoRI/KpnI restriction sites p3xFlag-CMV14-PI3K PI3K domain of DNA-PKcs at

N-terminus of 3xFlag tag

Sigma Aldrich - Generated in this study by inserting the PI3K to EcoRI/KpnI restriction sites

pEGFP-N1 MCS at N-terminus of EGFP tag (contains neomycin/kanamycin resistance gene)

Clontech

pEGFP-Survivin-N1 Survivin at N-terminus of EGFP tag

(contains neomycin/kanamycin resistance gene)

Provided by Chrysi Petraki –

Doctorate study (Petraki, 2014) / Dr.

Stephanie Hehlgans - University Hospital Frankfurt

pEGFP-Survivin S20D-N1 S20D mutated Survivin at N-terminus of EGFP tag

(contains neomycin/kanamycin resistance gene)

Provided by Chrysi Petraki –

Doctorate study (Petraki, 2014) / Dr.

Stephanie Hehlgans - University Hospital Frankfurt

pEGFP-Survivin W67A-N1 W67A mutated Survivin at N-terminus of EGFP tag

(contains neomycin/kanamycin resistance gene)

Generated in this study

pEGFP-Survivin S20D-W67A-N1

S20D and W67A mutated Survivin at N-terminus of EGFP tag

(contains neomycin/kanamycin resistance gene)

Generated in this study

pEGFP-Survivin ∆BIR-N1 BIR domain deleted Survivin at N-terminus of EGFP tag

(contains neomycin/kanamycin resistance gene)

Provided by Chrysi Petraki –

Doctorate study (Petraki, 2014) / Dr.

Stephanie Hehlgans - University Hospital Frankfurt

Table 2. Characteristics of oligonucleotides used for cloning and site-directed mutagenesis.

Oligo Sequence (5’ 3’) Tm (^oC) Manufacturer

S20A-Fw tcaaggaccaccgcatcgctacattcaagaactgg 79.27 Eurofins Genomics, Ebersberg S20A-Rev ccagttcttgaatgtagcgatgcggtggtccttga 79.27 Eurofins Genomics, Ebersberg S20D-Fw ctcaaggaccaccgcatcgatacattcaagaactggcc 78.97 Eurofins Genomics, Ebersberg S20D-Rev ggccagttcttgaatgtatcgatgcggtggtccttgag 78.97 Eurofins Genomics, Ebersberg W25A-Fw atctctacattcaagaacgcccccttcttggagggctgc 74.7 Eurofins Genomics, Ebersberg W25A-Rev gcagccctccaagaagggggcgttcttgaatgtagagat 74.7 Eurofins Genomics, Ebersberg F27A-Fw attcaagaactggcccgccttggagggctgcg 74.6 Eurofins Genomics, Ebersberg F27A-Rev cgcagccctccaaggcgggccagttcttgaat 74.6 Eurofins Genomics, Ebersberg L28A-Fw attcaagaactggcccttcgccgagggctgcgcctgca 78.1 Eurofins Genomics, Ebersberg L28A-Rev tgcaggcgcagccctcggcgaagggccagttcttgaat 78.1 Eurofins Genomics, Ebersberg E29A-Fw aagaactggcccttcttggccggctgcgcctgca 76.7 Eurofins Genomics, Ebersberg E29A-Rev tgcaggcgcagccggccaagaagggccagttctt 76.7 Eurofins Genomics, Ebersberg C31A-Fw ttcttggagggcgccgcctgcaccccgga 76.6 Eurofins Genomics, Ebersberg C31A-Rev tccggggtgcaggcggcgccctccaagaa 76.6 Eurofins Genomics, Ebersberg T34A-Fw gctgcgcctgcgccccggagcgg 81.67 Eurofins Genomics, Ebersberg T34A-Rev ccgctccggggcgcaggcgcagc 81.67 Eurofins Genomics, Ebersberg T34D-Fw gggctgcgcctgcgacccggagcggatg 81 Eurofins Genomics, Ebersberg

T34D-Rev catccgctccgggtcgcaggcgcagccc 81 Eurofins Genomics, Ebersberg D53A-Fw actgagaacgagccagccttggcccagtgtttctt 73 Eurofins Genomics, Ebersberg D53A-Rev aagaaacactgggccaaggctggctcgttctcagt 73 Eurofins Genomics, Ebersberg W67A-Fw ttcaaggagctggaaggcgccgagccagatgacga 75.3 Eurofins Genomics, Ebersberg W67A-Rev tcgtcatctggctcggcgccttccagctccttgaa 75.3 Eurofins Genomics, Ebersberg E76A-Fw atgacgaccccatagaggcccataaaaagcattcgtcc 72.7 Eurofins Genomics, Ebersberg E76A-Rev ggacgaatgctttttatgggcctctatggggtcgtcat 72.7 Eurofins Genomics, Ebersberg K79A-Fw cccatagaggaacataaagcccattcgtccggttgcgcttt 74.4 Eurofins Genomics, Ebersberg K79A-Rev aaagcgcaaccggacgaatgggctttatgttcctctatggg 74.4 Eurofins Genomics, Ebersberg T117A-Fw gccaagaacaaaattgcaaaggaagccaacaataagaa

gaaagaat

78.02 Eurofins Genomics, Ebersberg

T117A-Rev attctttcttcttattgttggcttcctttgcaattttgttcttggc 78.02 Eurofins Genomics, Ebersberg T117D-Fw gagccaagaacaaaattgcaaaggaagacaacaataag

aagaaagaatttgagg

78.96 Eurofins Genomics, Ebersberg

T117D-Rev cctcaaattctttcttcttattgttgtcttcctttgcaattttgttc ttggctc

78.96 Eurofins Genomics, Ebersberg

HEAT1-Fw cggaattcggcggcatggcgggctccggagccggt 74.3 Eurofins Genomics, Ebersberg HEAT1-Rev ggggtaccgtgtttataaccttgcacggtcc 70.8 Eurofins Genomics, Ebersberg FATC-Fw cggaattcggcggcatgctgaaaaaaggagggtcatgg 74.9 Eurofins Genomics, Ebersberg FATC-Rev ggggtaccgtcatccagggctcccatcct 73.7 Eurofins Genomics, Ebersberg PI3K-Fw cggaattcggcggcatggaacaccctttcctggtgaag 75.9 Eurofins Genomics, Ebersberg PI3K-Rev ggggtaccgtatttttccaatcaaaggagggc 69.5 Eurofins Genomics, Ebersberg

PI3K-ATM-Fw

cggaattcggcggcatggtggaccacacaggagaat 75.2 Eurofins Genomics, Ebersberg

PI3K-ATM-Rev

ggggtaccgtcctctgctgtaaatacaaagct 69.5 Eurofins Genomics, Ebersberg

Surv-Fw ggggtaccggcggcatgggtgccccgacgttgc 80.7 Eurofins Genomics, Ebersberg Surv-Rev cgggatcccgatccatggcagccagctgctc 76.1 Eurofins Genomics, Ebersberg

∆BIR-Fw1 catgggtgccccgacgttgccccctgcctggcagccctttct ct

82.5 Eurofins Genomics, Ebersberg

∆BIR-Rev1 ctagagagaaagggctgccaggcagggggcaacgtcgg ggcacccatggtac

83.6 Eurofins Genomics, Ebersberg

∆BIR-Fw2 gctctagatctgtcaagaagcagtttgaaga 65.5 Eurofins Genomics, Ebersberg

∆BIR-Rev2 cgggatcccgatccatggcagccagctgctc 76.1 Eurofins Genomics, Ebersberg

Table 3. Characteristics of sequencing primers for confirmation of mutations/deletions.

Oligo Sequence (5’ 3’) Tm (^oC) Manufacturer

pEGFPC1for gatcactctcggcatggac 58.8 Eurofins Genomics, Ebersberg pEGFPC1rev cattttatgtttcaggttcaggg 57.1 Eurofins Genomics, Ebersberg CMVfor cgcaaatgggcggtaggcgtg 65.7 Eurofins Genomics, Ebersberg

pEGFPN1rev gtccagctcgaccaggatg 61 Eurofins Genomics, Ebersberg

Table 4. Characteristics of siRNAs used for control or knockdown of endogenous Survivin expression.

siRNA Sequence (5’ 3’) Manufacturer Catalog

number AllStars Neg. Control

siRNA (20 nmol) - QIAGEN, Hilden 1027281

BIRC5 Survivin 3 siRNA

Sense: GCAGGUUCCUUAUCUGUCAtt Antisense: UGACAGAUAAGGAACCUGCag

Ambion, Austin,

TX, USA s1458

Table 5. Characteristics of primary antibodies used for immunoblotting, immunoprecipitation and immunofluorescence staining.

Target Host Type

Stock solution [µg/ml]

Dilution

Molecular weight

[kDa]

Manufacturer Catalog number

Anti-β-Actin mouse IgG 1000 1:10 000 42 Sigma-Aldrich A5441

Anti-Survivin rabbit IgG 200 1:1000 16.5 R&D Systems AF886

Anti- phospho-H2AX (Ser139)

mouse IgG1 1000 1:1000 17 Millipore 05-636

Anti-53BP1 rabbit IgG 1 1:1000 250 Novus

Biologicals NB100-304

Anti-FOXO3 rabbit IgG 15 1:1000 82-97 Cell Signaling 2497S

Anti-

phospho-FOXO3 (Ser253)

rabbit IgG 908 1:500 97 Cell Signaling 9466S

Anti-GFP rabbit IgG 2000 1:2000 27 Abcam ab290

Anti-FLAG rabbit IgG 84 1:1000 1 Cell Signaling 2368S

Anti-FLAG

(HRP) mouse IgG1 1100 1:1000 1 Abcam ab49763

Anti-DNA-PKcs mouse IgG 200 1:500 470 Thermo MS-370-P1

Isotype

control rabbit IgG 400 1:100 - Santa Cruz

Biotechnology SC-2027 Isotype

control mouse IgG 400 1:100 - Santa Cruz

Biotechnology SC-2025

Table 6. Characteristics of secondary antibodies used for immunofluorescence staining.

Target Host Type Dilution Label Manufacturer Catalog

number Anti-rabbit goat IgG 1:500 Alexa Fluor^R 488 Life technologies A11034 Anti-mouse goat IgG 1:500 Alexa Fluor^R 594 Life technologies A11032

Table 7. Characteristics of secondary antibodies used for immunoblotting.

Coupled enzyme Specificity Host Type Dilution Manufacturer Catalog number Horse radish peroxidase rabbit goat IgG 1:1000 Southern Biotech 4050-05 Horse radish peroxidase mouse goat IgG 1:1000 Southern Biotech 1030-05

Im Dokument Spatial and functional interrelationship of a heterotetramer Survivin-DNA-PKcs complex in the repair of DNA double-strand breaks (Seite 32-37)